Relationship of cyst nematode gene frequencies to soybean resistance

Summary Soybean (S, Glycine max (L.) Merr.) lines with relatively few cysts of soybean cyst nematode (CN, Heterodera glycines Ichinohe) populations are usually called CN-resistant. The phenotype of number of cysts per plant is of the CN-S (Cyst Nematode-Soybean) association and determined by the interactions of genes for avirulence-resistance. The acronym alins was proposed for these alleles for incompatibility, with xalin representing the interaction X of one microsymbiont malin with its host h-alin. These alins are dominant in the gene-for-gene model but may be mostly recessive with CN-S. Definitive genetic studies have been hindered by the heterogeneity of sexually reproducing CN populations and lack of the appropriate genetic models. Loegering's abstract interorganismal genetic model was modified so that one model represented all four possible interactions of dominant-recessive alins for an incompatible phenotype. This involved redefining the Boolean algebra symbol 1 to represent both the alins AND their frequencies. The model was used to derive the relationship: {ie893-01} where the expectation E of cysts (of any CN-S combination, as proportion of number of cysts on a check cultivar) is proportional to the product of CN genotypic frequencies expressed as functions of m-alin frequencies. Each m-alin is at a different locus, i.e., {ie893-02}. The number of terms multiplied for each CN-S is equal to the number of alins in the S line (or F₂ plant). There are too many unknowns in the equation to solve for any of them. The relationship does explain the continuous distributions of phenotypes that were nearly always observed. Basic genetic principles were used to concurrently derive the models and to obtain discontinuous distributions of numbers of cyst phenotypes in segregating generations due to one recessive alin in a CN-susceptible soybean line.Contribution from the Missouri Agricultural Experiment Station, Journal Series No. 9739     

Genetic mapping of soybean cyst nematode race-3 resistance loci in the soybean PI 437.654

Resistance to the soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is difficult to evaluate in soybean [Glycine max (L.) Merr.] breeding. PI 437.654 has resistance to more SCN race isolates than any other known soybean. We screened 298 F₆₇ recombinant-inbred lines from a cross between PI 437.654 and BSR101 for SCN race-3 resistance, genetically mapped 355 RFLP markers and the I locus, and tested these markers for association with resistance loci. The Rhg ₄ resistance locus was within 1 cM of the I locus on linkage group A. Two additional QTLs associated with SCN resistance were located within 3cM of markers on groups G and M. These two loci were not independent because 91 of 96 lines that had a resistant-parent marker type on group G also had a resistant-parent marker type on group M. Rhg ₄ and the QTL on G showed a significant interaction by together providing complete resistance to SCN race-3. Individually, the QTL on G had greater effect on resistance than did Rhg ₄, but neither locus alone provided a degree of resistance much different from the susceptible parent. The nearest markers to the mapped QTLs on groups A and G had allele frequencies from the resistant parent indicating 52 resistant lines in this population, a number not significantly different from the 55 resistant lines found. Therefore, no QTLs from PI 437.654 other than those mapped here are expected to be required for resistance to SCN race-3. All 50 lines that had the PI 437.654 marker type at the nearest marker to each of the QTLs on groups A and G were resistant to SCN race-3. We believe markers near to these QTLs can be used effectively to select for SCN race-3 resistance, thereby improving the ability to breed SCN-resistant soybean varieties.     

Development of SNP markers and haplotype analysis of the candidate gene for rhg1, which confers resistance to soybean cyst nematode in soybean

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe) is one of the most destructive pests in the cultivation of soybean (Glycine max (L.) Merr.) worldwide. Markers based on the SCN resistance gene will enable efficient marker-assisted selection (MAS). We sequenced the candidate gene rhg1 in six resistant and two susceptible soybean genotypes and identified 37 SNPs (single nucleotide polymorphisms) among the sequences, of which 11 were in the coding region. Seven of these 11 SNPs led to changes in the amino acid sequence of the gene. The amino acid sequence we obtained differs from the previously published one by a stretch of 26–27 amino acids. Six codominant allele-specific SNP markers based on agarose gel detection were developed and tested in 70 genotypes, among which occurred only nine different haplotypes. Two neutrality tests (Tajima’s D and Fu and Li’s F) were significant for the six SNP loci in the 70 genotypes, which is consistent with intensive directional selection. A strong LD pattern was detected among five SNPs except 2868T > C. Two SNPs (689C > A and 757C > T) formed one haplotype (689C-757C) that was perfectly associated with SCN resistance. The new allele-specific PCR markers located in the alleged sequence of the rhg1 candidate gene, combined with the microsatellite marker BACR-Satt309, will significantly improve the efficiency of MAS during the development of SCN-resistant cultivars.     

Interactions between iron-deficiency chlorosis and soybean cyst nematode in Minnesota soybean fields

Experiments were conducted in four commercial fields differing in severity of iron-deficiency chlorosis (IDC), and soybean cyst nematode (SCN) in Waseca and Lamberton, Minnesota to determine the interaction between the IDC and SCN. Each experiment was a randomized complete block with a factorial treatment design including 23 cultivars with or without traits of resistance to SCN, and IDC. The study illustrated the interactive effects of the two defensive traits on the diseases and soybean yields. IDC rating was higher in SCN-susceptible than SCN-resistant soybean, suggesting SCN infection increased IDC. Resistance to IDC apparently increased SCN reproduction due to better soybean plant growth. Yield response to the defensive traits depended on the disease pressures in a field. When both IDC and SCN were present in a field, deploying SCN-resistance was the best solution to the problems. However, SCN-resistance suppressed soybean yields when used in fields without the disease problems. IDC-resistance increased yield of SCN-susceptible cultivars, but it did not result in detectable yield benefit of SCN-resistant cultivars in SCN-infested sites. Effective use of the defensive traits for management of IDC and SCN requires specific knowledge of the disease problems present in a field. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U. S. Department of Agriculture and the University of Minnesota.     

QTL associated with horizontal resistance to soybean cyst nematode in Glycine soja PI464925B

Soybean cyst nematode (Heterodera glycines Ichinohe; SCN) is the primary disease responsible for yield loss of soybean [Glycine max (L.) Merr.]. Resistant cultivars are an effective management tool; however, the sources currently available have common resistant genes. Glycine soja Sieb. and Zucc., the wild ancestor of domesticated soybean, represents a diverse germplasm pool with known SCN resistance. The objectives of this research were to: (1) determine the genetic variation and inheritance of SCN resistance in a G. max (‘S08-80’) × G. soja (PI464925B) F _4:5 recombinant inbred line (RIL) population; and (2) identify and evaluate quantitative trait loci (QTL) associated with SCN resistance. Transgressive segregation for resistance was observed, although neither parent was resistant to the Chatham and Ruthven SCN isolates. Broad sense heritability was 0.81 for the Ruthven and 0.91 for the Chatham isolate. Root dry weight was a significant covariate that influenced cyst counts. One RIL [female index (FI) = 5.2 ± 1.11] was identified as resistant to the Chatham isolate (FI < 10). Seventeen and three RILs infected with Chatham and Ruthven isolates, respectively, had mean adjusted cyst counts of zero. Unique and novel QTL, which derived resistance from G. soja, were identified on linkage groups I, K, and O, and individually explained 8, 7 and 5% (LOD = 2.1–2.7) of the total phenotypic variation, respectively. Significant epistatic interactions were found between pairs of SSR markers that individually may or may not have been associated with SCN resistance, which explained between 10 and 15% of the total phenotypic variation. Best-fit regression models explained 21 and 31% of the total phenotypic variation in the RIL population to the Chatham and Ruthven isolates, respectively. The results of this study help to improve the understanding of the genetic control of SCN resistance in soybean caused by minor genes resulting in horizontal resistance. The incorporation of the novel resistance QTL from G. soja could increase the durability of SCN-resistance in soybean cultivars, especially if major gene resistance breaks down.     

Identification of QTLs associated with resistance to soybean cyst nematode races 2, 3 and 5 in soybean PI 90763

Soybean cyst nematode (SCN) is a major soybean pest throughout the soybean growing regions in the world, including the USA. Soybean PI 90763 is an important SCN resistance source. It is resistant to several SCN populations including races 2, 3 and 5. But its genetics of resistance is not well known. The objectives of this study were to: (1) confirm quantitative trait loci (QTLs) for resistance to SCN race 3 in PI 90763 and (2) identify QTLs for resistance to SCN races 2 and 5. QTLs were searched in Hamilton × PI 90763 F_2:3populations using 193 polymorphic simple sequence repeats (SSRs) covering 20 linkage groups (LGs). QTLs for resistance to SCN were identified on LGs A2, B1, E, G, J and L. The same QTL was suggested for resistance to different SCN races where their 1-LOD support intervals of QTL positions highly overlapped. The QTL on LG G was associated with resistance to races 2, 3 and 5. The QTL on LG B1 was associated with resistance to races 2 and 5. The QTL on LG J was associated with resistance to races 2 and 3. The QTLs on LGs A2 and L were associated with resistance to race 3. The QTL on LG E was associated with resistance to race 5. We conclude that LGs A2 and B1 may represent an important distinction between resistance to SCN race 3 and resistance to SCN races 2 and 5 in soybean.     

Identification of a new major QTL associated with resistance to soybean cyst nematode (Heterodera glycines)

Resistance of soybean [Glycine max (L.) Merr.] to cyst nematode (SCN) (Heterodera glycines Ichinohe), one of the most destructive pathogens affecting soybean, involves a complex genetic system. The identification of QTLs associated with SCN resistance may contribute to the understanding of such system. The objective of this work was to identify and map QTLs for resistance to SCN Race 14 with the aid of molecular markers. BC₃F_2:3 and F_2:3 populations, both derived from an original cross between resistant cv. Hartwig and the susceptible line BR-92–31983 were screened for resistance to SCN Race 14. Four microsatellite (Satt082, Sat_001, Satt574 and Satt301) and four RAPD markers (OPAA-11₇₉₅, OPAE-08₈₃₇, OPR-07₅₄₈ and OPY-07₂₀₃₀) were identified in the BC₃F_2:3 population using the bulked segregant analysis (BSA) technique. These markers were amplified in 183 F_2:3 families and mapped to a locus that accounts for more than 40% of the resistance to SCN Race 14. Selection efficiency based on these markers was similar to that obtained with the conventional method. In the case of the microsalellite markers, which identify homozygous resistant genotypes, the efficiency was even higher. This new QTL has been mapped to the soybean linkage group D2 and, in conjunction with other QTLs already identified for SCN resistance, will certainly contribute to our understanding of the genetic basis of resistance of this important disease in soybean. Received: 12 October 1999 / Accepted: 14 April 2000     

High-throughput genotyping for a polymorphism linked to soybean cyst nematode resistance gene Rhg4 by using TaqmanTM probes

An individual soybean breeder can generate over one hundred thousand new genotypes each year. The efficiency of selection in these populations could be improved if these genotypes were effectively screened with one DNA marker that identified an important gene, and if laboratory throughput was high and costs were low. Our aim was to develop a rapid genotyping procedure for resistance to the soybean cyst nematode. A high-throughput genotyping method was developed with fluorogenic probes to distinguish between two insertion polymorphisms in alleles of an AFLP marker that is located about 50 kbp from the Rhg4 gene candidate. The assay uses the 5 exonuclease activity of Taq polymerase in conjunction with fluorogenic probes for each allele. The method can be used for scoring the polymorphism in a recombinant inbred line population and for screening parent lines in a breeding program. The Taqman^TM method of determining genotype was accurate in 90% of scores in the RIL population compared to 95% accuracy with electrophoresis. Among 94 cultivars that are parents in our breeding program allele 2 that is derived from the sources of resistance to SCN was common in resistant cultivars (30 of 56) but rare in susceptible cultivars (3 of 38). Therefore, this method can be applied to automated large-scale genotyping for soybean breeding programs.     

Two simple sequence repeat markers to select for soybean cyst nematode resistance coditioned by the rhg1 locus

The soybean cyst nematode (SCN) (Heterodera glycines Inchinoe) is the most economically significant soybean pest. The principal strategy to reduce or eliminate damage from this pest is the use of resistant cultivars. Identifying resistant segregants in a breeding program is a difficult and expensive process which is complicated by the oligogenic nature of the resistance and genetic variability in the pathogen. Fortunately, resistance at one SCN-resistance locus, rhg1, is generally accepted as a necessity for the development of resistant genotypes using any source of resistance and when challenged by any SCN race. Thus, the development of SCN resistant cultivars would be expedited if an effective and rapid system were available to identify breeding lines carrying a resistance allele at the rhg1 locus. In this study we report two simple sequence repeat (SSR) or microsatellite loci that cosegregate and map 0.4 cM from rhg1. Allelic variation at the first of these loci, BARC-Satt309, distinguished most, if not all, SCN-susceptible genotypes from those carrying resistance at rhg1 derived from the important SCN-resistance sources ’Peking’, PI 437654, and PI 90763. BARC-Satt309 was also effective in distinguishing SCN resistance sources PI 88788 and PI 209332 from many, but not all, susceptible genotypes. BARC-Satt309 cannot be used in marker-assisted selection in populations developed from typical southern US cultivars crossed with the important resistance sources PI 88788 or PI 209332 because these genotypes all carry the identical allele at the BARC-Satt309 locus. A second SSR locus, BARC-Sat_168, was developed from a bacterial artificial chromosome (BAC) clone that was identified using the primers to BARC-Satt309. BARC-Sat_168 distinguished PI 88788 and PI 209332 from southern US cultivars such as ’Lee’, ’Bragg’ and ’Essex’. Both BARC-Satt309 and BARC-Sat_168 were used to assay lines from SCN-susceptible×SCN-resistant crosses and proved to be highly effective in identifying lines carrying rhg1 resistance from those carrying the allele for SCN susceptibility at the rhg1 locus. Received: 5 November 1998 / Accepted: 3 February 1999     

Association of RFLP markers with loci conferring broad-based resistance to the soybean cyst nematode (Heterodera glycines)

Soybean cyst nematode (SCN) is a major soybean yield-limiting pest. The present study was conducted to map broad-based SCN resistance loci from the cultivar Hartwig. Two-hundred F₂₃ lines derived from the cross Williams 82 x Hartwig were screened with a fourth-generation SCN inbred and 56 polymorphic molecular markers. Allele states and phenotypes were analyzed using stepwise regression and the model selection was made at P 0.01. Four unlinked RFLP markers (A006, A567, A487, A112) were associated with SCN resistance and the partial coefficient of determinations (R²) were 91%, 1%, 1%, and 1%. We have mapped a new, major SCN resistance locus (A006) and three minor loci (A567, A487, A112). This complete mapping will accelerate the transfer of broad-based resistance without linkage drag and aid in the determination of relationships among various SCN-resistant germplasm sources.     

SNP identification and marker assay development for high-throughput selection of soybean cyst nematode resistance

Background

Soybean cyst nematode (SCN) is the most economically devastating pathogen of soybean. Two resistance loci, Rhg1 and Rhg4 primarily contribute resistance to SCN race 3 in soybean. Peking and PI 88788 are the two major sources of SCN resistance with Peking requiring both Rhg1 and Rhg4 alleles and PI 88788 only the Rhg1 allele. Although simple sequence repeat (SSR) markers have been reported for both loci, they are linked markers and limited to be applied in breeding programs due to accuracy, throughput and cost of detection methods. The objectives of this study were to develop robust functional marker assays for high-throughput selection of SCN resistance and to differentiate the sources of resistance.

Results

Based on the genomic DNA sequences of 27 soybean lines with known SCN phenotypes, we have developed Kompetitive Allele Specific PCR (KASP) assays for two Single nucleotide polymorphisms (SNPs) from Glyma08g11490 for the selection of the Rhg4 resistance allele. Moreover, the genomic DNA of Glyma18g02590 at the Rhg1 locus from 11 soybean lines and cDNA of Forrest, Essex, Williams 82 and PI 88788 were fully sequenced. Pairwise sequence alignment revealed seven SNPs/insertion/deletions (InDels), five in the 6th exon and two in the last exon. Using the same 27 soybean lines, we identified one SNP that can be used to select the Rhg1 resistance allele and another SNP that can be employed to differentiate Peking and PI 88788-type resistance. These SNP markers have been validated and a strong correlation was observed between the SNP genotypes and reactions to SCN race 3 using a panel of 153 soybean lines, as well as a bi-parental population, F₅–derived recombinant inbred lines (RILs) from G00-3213 x LG04-6000.

Conclusions

Three functional SNP markers (two for Rhg1 locus and one for Rhg4 locus) were identified that could provide genotype information for the selection of SCN resistance and differentiate Peking from PI 88788 source for most germplasm lines. The robust KASP SNP marker assays were developed. In most contexts, use of one or two of these markers is sufficient for high-throughput marker-assisted selection of plants that will exhibit SCN resistance.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1531-3) contains supplementary material, which is available to authorized users.     

Loci underlying resistance to Race 3 of soybean cyst nematode in Glycine soja plant introduction 468916

Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is an important soybean [Glycine max (L.) Merr.] pest in the U.S. and throughout the world. Genetic resistance is the primary method for controlling SCN and there is a need to identify new resistance genes. Glycine soja Sieb. and Zucc. is the wild ancestor of domesticated soybean and is a potential source of new SCN resistance genes. The goal of this research was to map quantitative trait loci (QTLs) that provide resistance to SCN Race 3 from the G. soja plant introduction (PI) 468916. Fifty seven F₂-derived lines from a cross between the G. soja PI 468916 and the G. max experimental line A81-356022 were tested for resistance to an SCN population with a Race-3 phenotype. These lines were also genotyped with 1,004 genetic markers and resistance genes were mapped by composite interval mapping with the computer program QTL-Cartographer. In the F₂ population, three significant (LOD > 3.0) QTLs were detected that explained from 5% to 27% of the variation for Race-3 resistance. The two most significant QTLs identified in the F₂ population were tested in a population of 100 BC1F₂ plants developed by crossing A81-356022 to a line from the F₂ population that carried the two resistance QTLs from G. soja. In the backcross population, both Race-3 resistance QTLs were significant, which confirms the existence of these QTLs. The QTLs identified in this experiment map to positions where SCN resistance genes have not been previously identified, suggesting that these are novel genes that could be useful for diversifying the resistance genes currently used in cultivar development. Received: 7 August 2000 / Accepted: 4 December 2000     

Physical mapping of resistant and susceptible soybean genomes near the soybean cyst nematode resistance gene Rhg4.

The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is the foremost pest of soybean (Glycine max L. Merr.). The rhg1 allele on linkage group (LG) G and the Rhg4 allele on LG A2 are important in conditioning resistance. Markers closely linked to the Rhg4 locus were used previously to screen a library of bacterial artificial chromosome (BAC) clones from susceptible 'Williams 82' and identified a single 150-kb BAC, Gm_ISb001_056_G02 (56G2). End-sequenced subclones positioned onto a restriction map provided landmarks for identifying the corresponding region from a BAC library from accession PI 437654 with broad resistance to SCN. Seventy-three PI 437654 BACs were assigned to contigs based upon HindIII restriction fragment profiles. Four contigs represented the PI 437654 counterpart of the 'Williams 82' BAC, with PCR assays connecting these contigs. Some of the markers on the PI 437654 contigs are separated by a greater physical distance than in the 'Williams 82' BAC and some primers amplify bands from BACs in the mid-portion of the connected PI 437654 BAC contigs that are not amplified from the 'Williams 82' BAC. These observations suggest that there is an insertion in the PI 437654 genome relative to the 'Williams 82' genome in the Rhg4 region.     

Engineered resistance and hypersusceptibility through functional metabolic studies of 100 genes in soybean to its major pathogen, the soybean cyst nematode

During pathogen attack, the host plant induces genes to ward off the pathogen while the pathogen often produces effector proteins to increase susceptibility of the host. Gene expression studies of syncytia formed in soybean root by soybean cyst nematode (Heterodera glycines) identified many genes altered in expression in resistant and susceptible roots. However, it is difficult to assess the role and impact of these genes on resistance using gene expression patterns alone. We selected 100 soybean genes from published microarray studies and individually overexpressed them in soybean roots to determine their impact on cyst nematode development. Nine genes reduced the number of mature females by more than 50 % when overexpressed, including genes encoding ascorbate peroxidase, β-1,4-endoglucanase, short chain dehydrogenase, lipase, DREPP membrane protein, calmodulin, and three proteins of unknown function. One gene encoding a serine hydroxymethyltransferase decreased the number of mature cyst nematode females by 45 % and is located at the Rhg4 locus. Four genes increased the number of mature cyst nematode females by more than 200 %, while thirteen others increased the number of mature cyst nematode females by more than 150 %. Our data support a role for auxin and ethylene in susceptibility of soybean to cyst nematodes. These studies highlight the contrasting gene sets induced by host and nematode during infection and provide new insights into the interactions between host and pathogen at the molecular level. Overexpression of some of these genes result in a greater decrease in the number of cysts formed than recognized soybean cyst nematode resistance loci.